The microtubule cytoskeleton is essential to neuronal activity. Microtubules have an intrinsic polarity that motors read out to localize cargo, and differences in microtubule orientation between axons and dendrites are a defining feature of a polarized neuron (microtubule polarity is uniform in axons and mixed in dendrites). Despite the importance of microtubule organization to neuronal function, the mechanisms that create and maintain polarized microtubule arrays in axons and dendrites are poorly understood. While existing models focus largely on the motor-mediated translocation of microtubules into and out of neurites, there is now strong evidence from both vertebrates and invertebrates that local, non-centrosomal microtubule nucleation affects microtubule polarity in axons and dendrites, signaling the need for new models and a better understanding of local nucleation mechanisms. Our proposal addresses three fundamental, outstanding questions. How is nucleation machinery localized by molecular motors to specific compartments (Aim 1)? How is local ?-tubulin- mediated microtubule nucleation regulated to maintain the unique polarities of axonal and dendritic cytoskeletons, and how does local microtubule growth affect intracellular transport (Aim 2)? Using a fly model, we exploit cutting-edge genome engineering and live imaging approaches to dissect novel mechanisms of motor-based transport and local nucleation in vivo. There are two known platforms for ?-tubulin-mediated microtubule nucleation in neurons: Golgi outposts (dendrites only) and augmin (dendrites and axons). In Aim 1, we delineate a novel mechanism of polarized transport in which the coordinated and spatially regulated activities of kinesin-1 and dynein localize Golgi outposts to dendrites. In Aim 2, we determine how the localization of ?-tubulin to Golgi outposts or augmin (or novel nucleation centers) regulates microtubule polarity in axons and dendrites, and the effects of local microtubule growth on the transport of vesicles and organelles. To identify novel regulators of microtubule nucleation and microtubule polarity, we are leveraging our in vivo system in a forward genetic screen to gain new insights into the poorly understood mechanisms controlling local nucleation and microtubule polarity. Our studies will create a new mechanistic framework for understanding how polarized transport of Golgi outposts and local microtubule nucleation maintains neuronal polarity and supports intracellular trafficking. Multiple human disorders are associated with deficits in microtubule-based trafficking, and with mutations in kinesin-1 and dynein, and our investigations may shed light on the pathology of these diseases.